The Purification of a Nitro-reductase of Nocardia V.

Although several nit.ro-reductase systems, either from animals or microorganisms, have been identified in intact cells or cell-free homogenates, partial purification of the enzymes has been reported only for extract.s of Neurospora crassa (1) and Aspergillus niger (2, 3). Purification of the enzyme or enzymes involved in the reduction of organic nitro compounds is important for two reasons. (a) There is doubt whether the activity can be attributed to a single enzyme or a combination of enzymes. (b) As Nason (4) has reported, it is unknown whether the nitro-reductase is a specific system or whether the nitro compounds can serve as nonspecific electron acceptors for a number of enzyme systems. A further point of interest arises from the suggestion that the reduction of inorganic nitro compounds might take place after their transformation or incorporation into an organic form (5). Although later studies with Neurospora mutants have not supported this hypothesis, the possibility that an organic pathway exists for nitrate reduction has not been completely discarded. In order to study the properties of the purified nitro-reductase system and the mechanism of electron transfer, it was necessary to have a readily available source of crude material. Such a source is provided by a crude extract of Nocardia V. The present paper describes a scheme for the separation and purification of the Nocardia nitro-reductase enzyme. Ammonium sulfate fractionation in conjunction with ion exchange chromatography provide a convenient and rapid procedure for the isolation of nitro-reductase in highly purified form. The enzyme preparation so obtained may still be contaminated with other substances.